1. Field of the Invention
The present invention relates generally to the fields of neurobiology, pharmacology and psychiatry. More particularly, it concerns a screening method for identifying blockers or inhibitors of serotonin transport.
2. Description of Related Art
Neurotransmitters mediate signal transduction in the nervous system and modulate the processing of responses to a variety of sensory and physiological stimuli. An important regulatory step in neurotransmission is the inactivation of a neurotransmitter following its release into the synaptic cleft. This is especially true for the biogenic amine and amino acid neurotransmitters. Inactivation of neurotransmitters is typically mediated by uptake of the released neurotransmitter by neurotransmitters transporters that are located on the presynaptic neuron or in some cases on adjacent glial cells. Thus, neurotransmitter transporters are central to the processing of information in the nervous system and are associated with numerous neurological disorders.
For example, the neurotransmitter norepinephrine (also called noradrenalin) transduces signaling in the central nervous system that modulates attention, mood, arousal, learning, and memory (Aston-Jones et al., 1999; Coull et al., 1999; Skrebitsky and Chepkova, 1998; Hatfield and McGaugh, 1999). Norepinephrine (NE) transporters (NETs) attenuate neuronal signaling via rapid neurotransmitter clearance (Ressler and Nemeroff, 1999; Iversen et al., 1967; Axelrod and Kopin, 1969; Blakely et al., 1991). Norepinephrine transport is implicated in the pathology of major depression, post-traumatic stress disorder and attention deficit disorder (Ressler and Nemeroff, 1999; Southwick et al., 1999; Dow and Kline, 1997; Biederman and Spencer, 1999). Therapeutic agents that inhibit NET can elevate the concentration norepinephrine in the brain and periphery (Axelrod and Kopin, 1969; Bonisch, 1984; Ramamoorthy et al., 1993; Galli et al., 1995; Corey et al., 1994; Fleckenstein et al., 1999). Noradrenergic signaling in the peripheral nervous system influences blood pressure and heart rate (Jones, 1991; Jacob et al., 1999; Hartzell, 1980), and NET inhibitors, such as cocaine and antidepressants, induce cardiac complications (Watanabe et al., 1981; Clarkson et al., 1993; Glassman et al., 1985).
Similarly other neurotransmitters such as epinephrine (E), dopamine (DA), serotonin (SE), and their respective transporters such as epinephrine transporters (ET), dopamine transporters (DAT), and the serotonin transporters (SERT), mediate diverse aspects of neuronal signaling and are involved in the pathology of numerous nervous system related disorders. Thus, neurotransmitter transporters are the targets of various therapeutic agents used in the treatment of neurological disorders including, depression, epilepsy, schizophrenia, Parkinson's disease, attention deficit disorders, eating and sleeping disorders as well as some neurodegenerative disorders. In some instances, treatment of these disorders is mediated by the use of pharmaceutical agents that are antagonists of a neurotransmitter transporter. Antagonists block uptake and prolong and/or enhance the action of the neurotransmitter. For example, imipramine, a blocker of SE and NE uptake, is used as an antidepressant; benztropine, an antagonist of dopamine uptake, temporarily alleviates the symptoms of Parkinson's disease; and blockers of γ-amino butyric acid (GABA) uptake are used in the treatment of epilepsy.
Despite the relevance of neurotransmitter transporters, the art is hindered by very limited methods that are used in studying neurotransmitter transporter functions such as kinetics, affinity, temporal and spatial aspects of transport, voltage dependence and other transport mechanics (Galli et al., 1995; Corey et al., 1994; DeFelice and Galli, 1998; Prasad and Amara, 2001). Methods used to study neurotransmitter transport typically involve the use of radiometric substrates to measure neurotransmitter accumulation.
Unfortunately, traditional assays have made use of either native SERT or cloned SERT expressed in cells. These assays lack the ability assess whether the action of a drug is having its physiologic effect only by SERT blockade, even though it blocks the transporter in vitro or through other means as well. What is needed in the art is a novel form of SERT that lacks high affinity recognition of SSRIs to allow for discovery of novel drugs that block SERT in distinct ways and thus provide novel modes of action of SSRIs.
Thus, new methods for the analysis of neurotransmitter transport function are highly desirable in addition to cost effective and rapid screening methods to identify modulators of neurotransmitter transporters that may be useful as therapeutic agents in the treatment of nervous system disorders.